Traditional biochemical assays to detect intracellular activities, including immunoblotting and immunostaining, require the cell lysis or fixation, which can result in the alteration or loss of biologically relevant information. The application of fluorescence resonance energy transfer (FRET) can detect the intracellular signals in live cells with high spatiotemporal resolution, and hence provide more accurate and detailed information. FRET occurs when two fluorophores are in proximity, with the emission spectrum of the donor overlapping the excitation spectrum of the acceptor. Conventional FRET is based upon the cyan and yellow fluorescent protein pair (CFP and YFP, respectively). However, this biosensor only allows a single molecular activity to be visualized in live cells.
A variety of new fluorescence proteins with different colors have been developed (Shaner, et al. (2004) Nature Biotechnology 22:1567-1572). However, specific FRET pairs amongst these new fluorescent proteins are limited.
To simultaneously visualize more than one molecular event in the same cell, two spectrally distinctive pairs of fluorescence proteins capable of FRET are needed in the art. The present invention meets this need in the art.